The goal of this Bioengineering Research Grant (BRG) is to produce a novel multi-photon microendoscopy instrument that can rapidly vary focal depth and thus produce real-time vertical cross-sectional images from biological tissue in vivo, and to validate instrument performance in imaging of targeted peptide markers for colon cancer. Relative to prior miniature multi-photon instruments, the proposed instrument will feature much faster axial scanning for cross-sectional imaging without motion artifacts (frame rate 5-10 Hz) while maintaining a small diameter (3.4 mm) for compatibility with small animal imaging in the gastrointestinal tract. Instrument performance is expected to meet goals of sub-cellular resolution and deep tissue penetration with substantial field of view. Instrument development will be based on a multi-disciplinary collaboration of medical and engineering expertise in actuation, instrument design, optics, and cancer biology. Axial (into-tissue) scanning capabilities will be provided by high-speed, large- stroke thin-film PZT vertical translational actuators. A fiber-coupled, remote scanning architecture will permit modular insertion of optimized optics and actuators in a small handheld instrument. Instrument verification will be performed through benchmarking against of resolution, penetration depth, frame rate, and signal-to-noise ratio on standardized targets and/or phantom tissues. Full instrument validation will be performed in studies of in vivo imaging of genetically-engineered mouse models of cancer. Mice will be labeled with both targeted, dye-labeled peptides and general tissue dyes. Peptides will be optimized for bonding to specific targets exhibited in human disease and the mouse model. The optimized peptide will be characterized for binding affinity. In vivo imaging of the peptide with different fluorescent reporters will be used to identify optimal operating parameters for deep multi-photon imaging and characterize instrument performance.